Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.
Bottom Line: In ER- cases, the prognostic effect did not reach statistical significance.However, down-regulation of SPINK1 resulted in cell death.Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain.
Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.Show MeSH
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Mentions: Although we observed significant correlation of SPINK1 to DMFS mainly in ER+ patients, we investigated the mechanistic effects of SPINK1 in both ER+ and ER− cells. We attenuated SPINK1 transcript levels by siRNA in the breast cancer cell lines MCF-7 and MDA-MB-231 and conducted assays to detect phenotypic changes in cell proliferation, motility, invasiveness and anchorage-independent growth. Each of the 3 SPINK1-targeted siRNA constructs reduced SPINK1 levels in MCF-7 and MDA-MB-231 cells by >50%, with C2 siRNA being the most efficient (>95%; Fig 5A). Consistent with a threshold effect, C2 siRNA led to the most significant reduction of proliferation for both MCF-7 and MDA-MB-231 (Fig 5B), whereas, C1 and C3 siRNAs displayed marginal phenotypes. We posited that the observed decrease in proliferation could reflect either cell cycle arrest or apoptosis. Therefore, we studied the activation of apoptotic markers PARP and caspase-3 using a cell-based immunofluorescence assay. Attenuation of SPINK1 induced activation of both PARP and caspase-3 in MDA-MB-231 (p < 0.05, Fig 5C, left panel) and MCF-7 cells (Fig 5C, right panel). Secondly, since loss of SPINK1 leads to excessive autophagy in animal model systems, we studied if siRNA mediated knockdown of ATG5/12 was able to rescue cell death caused by loss of SPINK1. As shown in Fig S4 of Supporting information, interfering with autophagy could not rescue cell death. Taken together, the apparent decrease in cell proliferation upon loss of SPINK1 was due to the induction of cellular apoptosis. Therefore, SPINK1 appears to be essential for survival of breast cancer cells lines regardless of estrogen receptor status.
Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.